1 Volume 22 2023 e238998 Original Article Braz J Oral Sci. 2023;22:e238998http://dx.doi.org/10.20396/bjos.v22i00.8668998 1 Department of Stomatology, School of Dentistry, Federal University of Paraná, Curitiba, Paraná, Brazil. 2 Bone Marrow Transplantation Program, Clinical Hospital Complex, Federal University of Paraná, Curitiba, Paraná, Brazil. Corresponding author: Juliana Lucena Schussel DDS, PhD Department of Stomatology, Federal University of Paraná, Av. Lothário Meissner 632 Jardim Botânico Curitiba - Paraná - Brazil CEP: 80210-170 Telephone: +55 41 3360-4024 Fax: +55 41 33604053 E-mail: juliana.schussel24@gmail.com Editor: Dr. Altair A. Del Bel Cury Received: August 8, 2022 Accepted: November 11, 2022 Can NLR be a biomarker for mucositis and gvhd in patients undergoing allogeneic HSCT? Isabella Christina Costa Quadras1 , Fernanda Aparecida Stresser1 , Stephanie von Stein Cubas Warnavin1 , Vaneuza Araújo Moreira Funke2 , Rafael Zancan Mobile1 , Juliana Lucena Schussel1,* Allogeneic hematopoietic stem cell transplantation (HSCT) is a treatment for many diseases; however, it can induce complications such as Oral Mucositis (OM) and Graft-versus- Host Disease (GVHD). The neutrophil-lymphocyte ratio (NLR) is a peripheral biomarker of systemic inflammation and an independent prognostic factor for several inflammatory diseases. Aim: This study aimed to evaluate the association of NLR with OM and GVHD in patients undergoing allogeneic HSCT. Methods: Patients who underwent allogeneic HSCT at the Bone Marrow Transplant Service of the Hospital de Clínicas Complex of the Federal University of Paraná were included in the study. Socio-demographic data and blood counts were collected from patients’ medical records. The NLR was calculated and associated with OM and GVHD. Results: 45 patients were included in the study. Although NLR was higher in patients with OM and oral GVHD, no statistical difference was observed, and no relationship between OM and GVHD with NLR could be stated. Conclusion: Although both OM and GVHD are associated with an inflammatory response as well as the immune system, it was not associated with NLR. Further investigation considering other variables related to HSCT might find possible associations, as it could favor patient management and prevention. Keywords: Neutrophils. Lymphocytes. Hematopoietic stem cell transplantation. Mucositis. Graft vs host disease. https://orcid.org/0000-0003-0541-7254 https://orcid.org/0000-0002-7995-3466 https://orcid.org/0000-0001-9829-6230 https://orcid.org/0000-0002-2122-7277 https://orcid.org/0000-0002-8603-2614 https://orcid.org/0000-0001-5204-0782 2 Quadras et al. Braz J Oral Sci. 2023;22:e238998 Abbreviations HSCT, Hematopoietic stem cell transplantation; OM, Oral Mucositis; GVHD, Graft-ver- sus-Host Disease; NLR, Neutrophil-lymphocyte ratio; STMO-CHC, Serviço de Trans- plante de Medula Óssea of the Complexo Hospital de Clínicas; MASCC/ISOO, Multi- national Association of Supportive Care in Cancer and International Society of Oral Oncology; SPSS, Statistical Package for the Social Sciences. Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is considered a curative treatment for a variety of neoplastic and non-neoplastic hematological diseases1,2 that cause bone marrow defects, such as anemia, leukemia, and lymphoma1,3. HSCT occurs by replacing receptor cells with hematopoietic progenitor cells4. Oral mucositis (OM) and Graft-versus-Host Disease (GVHD) are potential conse- quences of HSCT. OM is an inflammation of the mucosa that occurs in approximately 75% of patients who receive ablative chemotherapy or total body irradiation as condi- tioning for HSCT, intensifying in the first two weeks after transplantation, which may reduce their ability to ingest food due to pain and discomfort5. The pathophysiology of GVHD is not yet fully understood6, however it is known to result from an immunological attack by donor immunocompetent T-cells in the recip- ient patient’s tissues7, either directly or through exaggerated inflammatory responses after allogeneic HSCT, manifesting in 30–50% of cases7-9. GVHD can affect one or more sites in the body and is considered one of the main causes of morbidity and mortality after HSCT. The oral cavity is frequently affected, especially chronic variant that correspond to the manifestations present after the +100 day after HSCT and affect between 25% and 83% of patients1,3,8,10. The neutrophil-lymphocyte ratio (NLR), initially described by Zahorec et al.11, is a peripheral biomarker of systemic inflammation and an independent prognostic factor in several inflammatory, cardiovascular diseases, and solid hematological neoplasms12-15. NLR is an indirect measure of the imbalance between the innate immune (neutrophils) and adaptive or humorous (lymphocytes) systems, obtained by the absolute count of neutrophils divided by the absolute count of lymphocytes, which is a biomarker of low cost, reliability, and simple collection through a com- mon peripheral blood count12,14,16,17. It can be used in a series of inflammatory dis- eases12,17-22 and is an important marker of poor prognosis, overall survival, and dis- ease-free survival19. To the best of our knowledge, there is little evidence of the use of NLR as a biomarker after HSCT23, and perhaps no evidence of its application in the assessment of OM and GVHD after HSCT. OM and GVHD are exacerbated inflammatory processes that affect the prognosis of allogeneic HSCT patients. Therefore, it is essential to consider markers that can assist in the prediction, management, and treatment of these conditions. 3 Quadras et al. Braz J Oral Sci. 2023;22:e238998 Aim Considering the applicability of NLR to a series of inflammatory diseases, this study aimed to evaluate the association of NLR with OM and GVHD in patients undergoing allogeneic HSCT. Materials and Methods This longitudinal observational study included a convenience sample of 45 patients, older than 18 years, admitted to the Serviço de Transplante de Medula Óssea of the Complexo Hospital de Clínicas of the Universidade Federal do Paraná, and who under- went allogeneic HSCT. This study was approved by the Research Ethics Committee of the Complexo Hos- pital de Clínicas of the Universidade Federal do Paraná (number 4.414.355). Those who agreed to participate signed an informed consent form. The inclusion criteria consisted of patients older than 18 years who underwent HSCT. Exclusion criteria consisted of patients undergoing autologous HSCT, and those with Fanconi Ane- mia as an underlying disease were excluded. Sociodemographic data and blood count results were collected from patients’ medical records. NLR was calculated using a spreadsheet in Excel for Windows software. Oral mucositis (OM) and graft versus Host Disease (GVHD) were assessed by physical examination and classi- fied according to the MASCC/ISOO guidelines 202024 and NIH 2014 classifications, respectively25. Patients were evaluated pre-HSCT and 15, 30, 60, 90, 120, 150, and 180 days after HSCT. NLR data for association with OM were collected from the blood counts at 15 days post-HSCT, since this is the time when there is the most severe manifestation. For GVHD, patients were followed up monthly for 180 days after HSCT. NLR data for association with GVHD were collected from blood counts corresponding to the day of GVHD diagnosis, whereas NLR data from patients who did not manifest GVHD were collected from the last follow-up visit 180 days after HSCT. All patients underwent dental consultation and had oral cavity adequacy before the chemotherapy regimen. All patients received photobiomodulation to pre- vent OM from the first day of the conditioning regimen, following the MASSC/ISSO clinical practice guidelines. Statistical analysis was performed using SPSS version 20.0 (IBM, Armonk, New, USA). Results The 45 patients were mostly male (61.7%), with an average age of 37 years. The most frequent underlying disease was Severe Aplastic Anemia (31.9%), followed by Acute Myeloid Leukemia (29.8%); 66% of donors were related, and 55.3% were matched. Men had a higher frequency of OM than women. The most common underlying dis- ease is Severe Aplastic Anemia. Most allogeneic HSCT cases were related, and those who found an unrelated donor with a good match almost entirely showed OM. Most patients manifested OM regardless of graft compatibility. Regarding the OM grade, six cases were classified as grade 0; three as grade 1; eight as grade 2, and 14 as grades 3 and 4. 4 Quadras et al. Braz J Oral Sci. 2023;22:e238998 There was no statistically significant difference in OM associated with sex, underlying disease, donor relation, match, age, or NLR. The distribution of the sample according to sex, underlying disease, donor relation, and match associated with the presence or absence of OM is described in Table 1 below. Table 1. Distribution of clinical data and NLR associated with Oral Mucositis. VARIABLES ORAL MUCOSITIS TOTAL P VALUE YES NO Sex - n(%) 0,658* Female 14(82,4) 3(17,6) 17(100) Male 25(89,3) 3(10,7) 28(100) Total 39(86,7) 6(13,3) 45(100) Underlying disease - n(%) Acute lymphoid leukemia 8(100) 0(0) 8(100) - Acute myeloid leukemia 13(92,9) 1(7,1) 14(100) Non-Hodgkin’s lymphoma 2(50) 2(50) 4(100) Myelodysplastic syndrome 3(100) 0(0) 3(100) Severe aplastic anemia 11(78,6) 3(21,4) 14(100) Others 2(100) 0(0) 2(100) Total 39(86,7) 6(13,3) 45(100) Donor relation - n(%) Related 25(83,3) 5(16,7) 30(100) Unrelated 14(93,3) 1(6,7) 15(100) 0,647* Total 39(86,7) 6(13,3) 45(100) Match- n(%) Matched 22(88) 3(12) 25(100) Unmatched 17(85) 3(15) 20(100) 1,000* Total 39(86,7) 6(13,3) 45(100) Age – Median (min-max) 41(20-62) 24,50(19-60) - 0,300** NLR – Median (min-max) 1,13(0,00-22,45) 0,44(0,0027,70) - 0,917** * Fisher’s exact test. ** Mann Whitney U test. Regarding oral GVHD, the majority of the patients were men. The most frequent under- lying hematological disease was Severe Aplastic Anemia, followed by Acute Myeloid Leukemia. As for the donor relationship for allogeneic HSCT, most were related and matched. Over 30% (10/31) of patients developed oral manifestations of GVHD. Regarding the NLR, it was analyzed that the result obtained was greater in patients who had oral GVHD compared to those who did not. 5 Quadras et al. Braz J Oral Sci. 2023;22:e238998 For non-oral GVHD, similar results were found; a large part of the sample was men, mostly diagnosed with Severe Aplastic Anemia, with a related and matched donor in most cases. NLR was considerably higher in individuals who developed non-oral GVHD than in those who did not. However, no statistically significant difference was observed for oral and non-oral GVHD in terms of sex, underlying disease, donor relation, match, age, and NLR, except for the donor relation in non-oral GVHD, as shown in Tables 2 and 3. Table 2. Distribution of clinical data and NLR associated with Oral GVHD. VARIABLES ORAL GVHD TOTAL P VALUE YES NO Sex - n(%) 0,262* Female 2(18,2) 9(81,8) 11(100) Male 8(40) 12(60) 20(100) Total 10(32,3) 21(67,7) 31(100) Underlying disease - n(%) Acute lymphoid leukemia 1(33,3) 2(66,7) 3(100) - Acute myeloid leukemia 4(40) 6(60) 10(100) Non-Hodgkin’s lymphoma 2(66,7) 1(33,3) 3(100) Myelodysplastic syndrome 1(50) 1(50) 2(100) Severe aplastic anemia 2(16,7) 10(83,3) 12(100) Others 0(0) 1(100) 1(100) Total 10(32,3) 21(67,7) 31(100) Donor relation - n(%) Related 9(37,5) 15(62,5) 24(100) Unrelated 1(14,3) 6(85,7) 7(100) 0,379* Total 10(32,3) 21(67,7) 31(100) Match- n(%) Matched 7(36,8) 12(63,2) 19(100) Unmatched 3(25) 9(75) 12(100) 0,697* Total 10(32,3) 21(67,7) 31(100) Age – Median (min-max) 44,5(21-62) 32(19-60) - 0,526** NLR – Median (min-max) 2,63(0,43-14,50) 1,42(0,00-10,99) - 0,310** * Fisher’s exact test. ** Mann Whitney U test. 6 Quadras et al. Braz J Oral Sci. 2023;22:e238998 Table 3. Distribution of clinical data and NLR associated with Non-Oral GVHD. VARIABLES NON-ORAL GVHD TOTAL P VALUE YES NÃO Sex - n(%) 0,139* Female 2(18,2) 9(81,8) 11(100) Male 10(47,6) 11(52,4) 21(100) Total 12(37,5) 20(62,5) 32(100) Underlying disease - n(%) Acute lymphoid leukemia 2(66,7) 1(33,3) 3(100) - Acute myeloid leukemia 5(50) 5(50) 10(100) Non-Hodgkin’s lymphoma 3(75) 1(25) 4(100) Myelodysplastic syndrome 0(0) 2(100) 2(100) Severe aplastic anemia 2(16,7) 10(83,3) 12(100) Others 0(0) 1(100) 1(100) Total 12(37,5) 20(62,5) 32(100) Donor relation - n(%) Related 12(48) 13(52) 25(100) Unrelated 0(0) 7(100) 7(100) 0,029* Total 12(37,5) 20(62,5) 32(100) Match- n(%) Matched 8(42,1) 11(57,9) 19(100) Unmatched 4(30,8) 9(69,2) 13(100) 0,713* Total 12(37,5) 20(62,5) 32(100) Age – Median (min-max) 45(21-62) 32(19-60) - 0,520** NLR – Median (min-max) 3,19(0,43-5,69) 1,43(0,00-14,50) - 0,302** * Fisher’s exact test. ** Mann Whitney U test. Discussion NLR is a simple parameter that assesses a subject’s inflammatory state. It has been proven to be a strong prognostic factor in several types of cancer, major cardiac events, and markers of inflammation, infectious diseases, and postoperative compli- cations26,27. Therefore, this study aimed to evaluate the association of NLR with OM and GVHD in patients undergoing allogeneic HSCT. No association was observed between OM, GVHD, and NLR. Considering the association of NLR with inflammatory diseases and that OM and GVHD are also associated with the activity of inflammatory cells, we hypothesized that NLR could serve as a biomarker for these manifestations. Nonetheless, to the best of our knowledge, this is the first study to associate NLR with complications related to allogeneic HSCT. 7 Quadras et al. Braz J Oral Sci. 2023;22:e238998 Lesions in oral tissues can serve as an entry vehicle for the spread of bacterial, fungal and viral infections, especially in patients undergoing myelosuppressive or immuno- suppressive chemotherapy regimens for cancer treatment28. These patients develop oral problems 2 to 3 times more often than patients undergoing treatment for solid tumors. The results of the present study corroborate this statement, since the vast majority of patients with malignant blood diseases manifested OM (86.7%), a prog- nostic biomarker can help in the management of the condition. There is evidence that patients undergoing allogeneic HSCT develop OM more often and more severely than autologous transplant recipients12. But to the best of our knowledge, no studies have compared related and unrelated grafts in allogeneic HSCT. In the present study, related HSCT constituted the majority of allogeneic trans- plants (66.66%), which explains the large number of patients with OM who received HSCT from a related donor compared to unrelated HSCT. We did not find any statistically significant difference associated with gender when evaluating patients who developed oral and non-oral GVHD, as well as with sex. How- ever, when HSCT occurs from a female donor to a male host, the risk of GVHD devel- opment increases29. The most common underlying disease in patients with manifestations of oral and non-oral GVHD after allogeneic HSCT was Acute Myeloid Leukemia (AML), cor- responding to 40% of oral GVHD and 50% of non-oral GVHD. This result corrob- orates with others29. Carlens et al.29 (1998) suggests that AML is a risk factor for acute GVHD. Although we did not observe a statistically significant difference in terms of age, donor relationship, and match in oral and non-oral GVHD, a lower frequency was observed in individuals who were younger, had a related donor, and received a matched trans- plant. These data reaffirm the results of other studies by indicating that the age of the recipient and donor, the mismatch of human leukocyte antigens (HLA) or HLA disparity between donor and recipient, and unrelated donors are clinical risk factors that increase the chance for the development of GVHD3,29. According to Bassim et al.8 (2015), patients undergoing transplantation with unrelated donors are at a risk of up to 80% to develop GVHD8. An exception was the donor relationship in non-oral GVHD, in which a statistically significant difference was observed (p = 0.029), since none of the patients who received the unrelated transplant developed GVHD. However, these data contradict the results of other studies3,29 that show that GVHD occurs more frequently as a result of unrelated donors. We believe that our results do not indicate causality in this sample, since the small number of patients with manifestations of oral and non-oral GVHD can be explained by the fact that a large part of the sample was composed of young adults and the majority of the individuals received related and matched transplants. In our sample, we did not find a statistically significant difference in NLR associated with OM, oral GVHD, and non-oral GVHD. To the best of our knowledge, this is the first study to analyze this association. The search for biomarkers with easy access, low cost, and that can provide important answers in the diagnosis, treatment, and 8 Quadras et al. Braz J Oral Sci. 2023;22:e238998 prognosis of consequent manifestations of allogeneic HSCT is of paramount rel- evance. Finding a larger sample, in which NLR could be paired by age, underlying disease, conditioning regime, donor relation, and match between patients who develop OM, oral GVHD, and non-oral GVHD, is a great challenge; for this reason, we believe that future multicenter studies can help infer the results of the NLR associations with OM, oral GVHD, and non-oral GVHD for the general population of patients undergoing allogeneic HSCT. In conclusion, although both OM and GVHD are associated with the inflammatory response as well as the immune system, they are not associated with NLR. Declarations of interest None. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Ethics approval All procedures carried out in studies involving human participants were in accordance with the ethical standards of the institutional research committee and the Helsinki Declaration of 1964 and its subsequent amendments or comparable ethical stan- dards. The study was approved by the Research Ethics Committee of the Complexo Hospital de Clínicas of the Universidade Federal do Paraná (No. 4.414.355). Consent to participate Free and informed consent was obtained from all individual participants included in the study. Consent for publication Free and informed consent was obtained from all individual participants included in the study. Author Contribution • Isabella Christina Costa Quadras: Conceptualization, Methodology, Data curation, Writing - Original draft preparation. • Fernanda Aparecida Stresser: Conceptualization, Methodology, Data curation, Writing - Original draft preparation. • Stephanie Von Stein Cubas Warnavin: Conceptualization, Methodology, Data cu- ration, Writing - Original draft preparation, Review & Editing. • Sandra Regina da Silva: Conceptualization, Writing - Review & Editing. • Vaneuza Araújo Moreira Funke: Conceptualization, Writing - Review & Editing. • Rafael Zancan Mobile: Conceptualization, Methodology, Data curation, Writing - Original draft preparation, Review & Editing. 9 Quadras et al. Braz J Oral Sci. 2023;22:e238998 • Juliana Lucena Schussel: Supervision, Conceptualization, Methodology, Data cu- ration, Writing - Review & Editing. All authors actively participated in the manuscript’s findings, revised, and approved the final version of the manuscript. References 1. 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